JP2752201B2 - Electrical analysis method - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an electric analysis method, and in particular, to an electric analysis method capable of absolutely quantifying a substance to be determined based on a change in a voltage, a current, or an amount of electricity. It is about.

[Conventional technology]

Electrochemical analysis has recently been used in so-called biosensors and liquid chromatograph detectors, and the field of application has been expanded. As a detector using this method, an electrode that is a detection unit is immersed in a sample liquid. And those in which a sample solution is forced to flow in a detection electrode, and those in which a sample is directly introduced into an electrolytic solution.

[Problems to be solved by the invention]

However, when the electrolyte flows in the detection electrode in electroanalysis or when the interference component is not sufficiently masked, the background level increases or the S / N
The ratio tends to decrease, and in some cases, the above analysis methods may not provide sufficient detection sensitivity and accuracy.

In recent years, expectations for electrochemical analysis have been high, and there has been a strong demand for shortening the analysis time and improving the analysis accuracy.

An object of the present invention is to solve the above-mentioned problems of the prior art and to provide an electric analysis method having a short response time, high accuracy, and excellent operability.

[Means for solving the problem]

The present inventors have conducted intensive studies in an attempt to improve the above-mentioned disadvantages of the conventional technique, and as a result, by applying a permeable membrane to the detection electrode of the detector for electroanalysis, the sample is placed in the detection electrode in continuous fluid analysis. In the analysis in which the sample is administered, there is also an effect of removing interfering components by a filter effect, so that the background level of the detector is reduced, and the S / N ratio is improved, The present inventors have found that high-sensitivity, high-precision analysis can be performed quickly, and have reached the present invention.

That is, the present invention provides a method of electrolyzing a porous body of a detection electrode chamber in an electrolytic cell in which a detection electrode chamber having a detection electrode made of a conductive porous body and a counter electrode chamber having a counter electrode are adjacent to each other via an ion-permeable diaphragm. The sample is impregnated with the solution, and a certain amount of the sample that has passed through the permeable membrane is supplied and electrolyzed. By measuring at least one of the voltage, current, and electric quantity at the detection electrode, the amount of the sample in the sample is determined. It is characterized by absolute quantification of substances.

In electrical analysis, the amount of electrical change when a substance to be measured is electrolyzed, that is, the change in voltage, current, or amount of electricity is related to the amount of the electrolyzed substance, so the amount of electrical change during electrolysis is measured. By doing so, the substance to be quantified can be quantified.

The detector for electrical analysis used in the present invention uses an ion-exchange membrane, a microporous membrane, or the like as an ion-permeable diaphragm. In order to reduce the background level and perform stable quantification, it is preferable to use an ion-exchange membrane, but when it is necessary to greatly reduce the electric resistance of the detector, a simple porous membrane is preferable. .

As a detection electrode material, an element heavier than carbon is
Surface element number ratio measured by ESCA (photoelectron spectroscopy)
It is preferable to use an aggregate of carbonaceous or graphite carbon fibers or porous carbon which is present at 1.5% or more. The element coexisting on the electrode surface is introduced to the detection electrode surface as an oxide or carbide of an element such as a group V, VI, or VII element of the periodic table. As a result, the dispersibility of the sample is improved, and the electron transfer reaction between the detection electrode and the substance to be electrolyzed in the electrolytic reaction is rapidly performed.

At the detection electrode of the detector, the lead wire is usually taken out from the metal net for current collection. However, in the analysis in which only a relatively small current flows, the detection electrode is extended as it is to be taken out of the cell and collected. A current collector or an array of carbon fibers may be used as a current collecting cloth and a lead wire.

The permeable membrane that allows the sample to be supplied to the detection electrode chamber to pass therethrough is a specific component permeable membrane, and is used properly depending on the substance to be measured.
When the substance to be quantified is a liquid sample such as blood, the permeable membrane is, for example, a porous filter membrane, a filter membrane with a protein removing function, a permeable membrane, or the like.In the case of a gas sample and some liquid samples, for example, a semipermeable membrane, An ion exchange membrane, a cellulose filtration membrane, a synthetic resin membrane or the like is used.

In the detector for electric analysis used in the present invention, when a fixed-volume sample chamber is provided adjacent to the detection electrode via the permeable membrane, it is preferable to provide an inlet and an outlet for the sample in the sample chamber. As a result, the sample can be continuously supplied and the substance to be quantified in the sample can be continuously analyzed, or the continuous supply can be temporarily stopped and the absolute quantification for calibration can be performed in a stationary state.

In the present detector for electrical analysis, the distance between the permeable membrane and the wall surface of the sample chamber facing the permeable membrane (hereinafter, sometimes referred to as the height of the sample chamber) is within 3 mm, preferably 2 mm. When the interval is wider than 3 mm, the detection sensitivity is lowered and the calibration stability is often deteriorated.

Table 1 shows the detector sensitivity and the calibration stability when the residual chlorine in tap water and ozone in gas were determined by changing the height of the sample chamber of a detector having a circular cross section of 50φ. It shows.

By the way, in the case of analysis in which the required time is simply long and the system is stable and has small variations, the height of the sample chamber is set to 4 mm.
I can't help even with the above. In this case, the time required for analysis can be reduced by the method described below.

That is, the target component in the sample can be absolutely quantified by extrapolation of a plot of the current flowing through the detection electrode and the amount of electricity, or extrapolation of the logarithm of the detection electrode current and time of the blot. This method is a method called an i (current) -Q (electric quantity) plot or a logi-t (time) plot. When the current decreases exponentially with time, the plot becomes a straight line. . The quantity of electricity extrapolated to the point where the current has dropped to the level of the background current (the time integral of the current Q = ∫i ₀ · also in the logi-t plot)
^Find Q by e− ^αt dt, where i: initial bulb, α:
Absolute quantification of the target component can be performed using the coefficient. If this extrapolation method is applied to the method of the present invention, the analysis time can be as short as 2-3 minutes even when it takes as long as 15-20 minutes to complete the electrolysis. In this way, it is also possible to repeat the sample exchange in the sample chamber and the electrolysis for several minutes to intermittently perform the absolute quantification.

Examples of the shape of the sample chamber of the present detector include, but are not limited to, a cylindrical body, a cubic body or a rectangular parallelepiped, or a shape in which these are combined.

The electroanalytical method of the present invention using the above-described electroanalytical detector includes a sample in which a detection electrode is impregnated with an electrolytic solution, that is, a sample in which the electrolytic solution is stationary and a specific substance is passed through a permeable membrane. Is electrolyzed to perform absolute quantification. By electrolyzing the sample that has passed through the permeable membrane, only the specific substance, which is the target component, is quantified, so that the influence of the interfering substance can be avoided. Also, by making the electrolyte non-fluid state,
The background is extremely small and stable, and analysis sensitivity and analysis accuracy are improved.

Examples of the electrolyte used in the present invention include various pH buffers and the like. Examples thereof include redox mediators such as potassium iodide (metal oxide ions such as molybdate ions, ferricyan ions / Complex compounds such as ferrocyanide ions and iron aminocarboxylates),
What dissolved or dispersed the enzyme etc. can also be used.

When the sample is a liquid in the electroanalytical method of the present invention,
It can also be administered by dropping a fixed amount of the sample on the permeable membrane using a quantitative pipette or the like. At this time, the sample chamber of the detector is not required.

When it is necessary to continuously analyze a target component in a liquid sample or a gas sample, an electroanalytical detector having a sample chamber having an inlet and an outlet for the sample is used. In this case, the sample is continuously introduced at a predetermined flow rate from an inlet provided in the sample chamber of the detector, and the target component in the sample passes through the permeable membrane and continuously reaches the detection electrode. It is measured by voltammetry or amperometry. The sample after measurement flows out from the sample outlet of the sample chamber at a predetermined flow rate. That is, according to this analysis method, while the sample is flowing in the sample chamber, part or all of the target component in the sample is introduced into the detector through the permeable membrane, and is continuously quantified. In this case, temporarily stop the flow of the sample during continuous analysis, measure the change in current, electric quantity or voltage based on this, and calibrate the measurement value during continuous supply by obtaining the concentration of the substance to be determined. Can be.

In the electroanalytical method of the present invention, when the analyte is absolutely quantified, a sample is temporarily held in the sample chamber,
The measurement is performed by measuring a change in the amount of electricity or current during that time. In this case, the sample supply pump is stopped, and measures such as closing the inlet and outlet of the sample chamber by means such as a valve or a valve are taken. A capillary may be used instead of the valve. According to this absolute quantification method, even if the substance to be quantified in the sample chamber is not completely reacted, the current-
As described above, if an electric quantity curve or a log (current) -time curve is obtained, absolute quantification can be performed by extrapolated values.

In the electroanalytical method of the present invention, when the substance to be determined in the sample solution does not cause a direct electron transfer reaction with the detection electrode, a redox mediator such as iodine / iodine ion or an enzyme such as an enzyme described above is contained in the electrolytic solution. It is preferable that an auxiliary substance coexist. Since these auxiliary substances can convert a substance to be determined into an electrode reaction active substance, the oxidation-reduction mediators include halogen ions / halogens, hydroquinones / quinones (for example, naphthoquinonesulfonic acid, etc.), and polyvalent. Metal ions, etc., and as enzymes,
For example, a substance that generates hydrogen peroxide by reacting with a substance to be determined, such as glucose, or consumes oxygen, and another substance that generates an active material for a detection electrode such as oxygen directly or through a redox mediator. There is.

〔Example〕

FIG. 1 is a sectional view showing an example of a detector for electrical analysis used in the present invention. This detector for electrical analysis is a diaphragm 1
And a detection electrode chamber 3 having a detection electrode 2 and a counter electrode chamber 5 having a counter electrode 4 provided on both sides of the diaphragm 1 respectively.
And a permeable membrane 7 provided on the sample input surface of the detection electrode chamber 3
And a sample chamber 8 adjacent to the detection electrode chamber 3 via the permeable membrane 7, a capillary 9 provided in the sample chamber 8 for inflow and outflow of a sample, and a sample pump 10. Reference numeral 6 denotes a lead wire extracted from the detection electrode 2 or the counter electrode 4.

In such a configuration, the target substance in the sample introduced into the sample chamber 8 through the capillary 9 by the sample pump 10 is administered to the detection electrode 2 of the detection electrode chamber 3 through the permeable membrane 7 and electrolyzed. Is extracted through the lead wire 6 to determine the voltage, current or amount of electricity. on the other hand,
The sample from which the substance to be determined has been removed is discharged out of the sample chamber 8 through the capillary 9 in the direction opposite to the inlet.

Next, the present invention will be described in more detail with reference to specific examples.

Example 1 An actyl cellulose-based thin film having a thickness of 0.025 mm was used as the permeable membrane 7, and cholesterol oxidase (3
pH citrate buffer solution containing β-hydroxysteroid oxidase) and potassium ferrocyanide (0.3M) was used as a detection electrode and boiled in 1: 1 nitric acid for about 15 minutes to perform surface oxidation treatment. Cholesterol in blood was quantified using a 20 mm, 3 mm thick carbon chamber carbon felt detector with the sample chamber portion of the detector for electroanalysis shown in FIG. 1 omitted. All quantification results in the ten analyzes fall in the range of 170-185 mg / dl. The reproducibility was very good. The time required for one analysis was short, each about 30 seconds. The analysis value by the cholesterol measurement kit was 180 mg / dl.

According to the present embodiment, the test blood is directly transferred to the permeable membrane 7 by using an acetylcellulose-based thin film as the permeable membrane.
Even when the solution was applied above, only the serum portion was transferred to the detection electrode 3 and analyzed by decomposition, so that pretreatment such as centrifugation in the conventional method was not required, and cholesterol could be quantified quickly in a short time.

Comparative Example 1 Cholesterol in blood was quantified 10 times in the same manner as in Example 1 except that the test blood was directly administered to the detection electrode 2 without using the permeable membrane 7. The first quantification time was as short as about 20 seconds, but became longer as the number of times increased, and soon exceeded 1 minute. In addition, the quantitative values varied greatly, and the range was 175 to 230 mg / dl.

Example 2 Quantification of uric acid in urine was continuously performed 20 times in the same manner as in Example 1 except that a pH buffer solution containing uricase as an enzyme and calium ferrocyanide as a redox mediator was used as an electrolytic solution. As a result, stable analytical values of 6 to 8 mg / dl were obtained. The time for one analysis is 45 ~
50 seconds.

Comparative Example 2 The same quantification was performed in the same manner as in Example 2 except that the permeable membrane 7 was omitted, but the range of the quantification value varied from 4 to 9 mg / dl, and a stable result was not obtained.

Example 3 As an electrolytic solution, a pH in which potassium ferrocyanide was dissolved was used.
When ascorbic acid in natural fruit juice (orange), which is a reducing substance, was electrolyzed 10 times continuously in the same manner as in Example 1 except that a buffer solution was used, all values were 2.71 to 2.92 m-cron and stable values were obtained. Obtained. In addition, each analysis time was within 60 seconds.

Comparative Example 3 A similar analysis was performed in the same manner as in Example 3 except that the permeable membrane 7 was eliminated. As a result, the shape of the current peak became irregular and the fluctuation of the background level (dark current) became large. No good results were obtained.

Example 4 The sample flow path in the sample chamber 8 of the detector shown in FIG.
By means of a detector having a meandering channel as shown in FIG.
, A continuous quantification of residual chlorine in tap water was performed using a polyvinylidene chloride thin film and a 3N-acetic acid aqueous solution containing potassium iodide as an electrolyte. The flow of the sample in the sample chamber 8 was temporarily stopped, and a decrease in the current value was observed for one minute, an iQ curve (i: current, Q: electric quantity) was created, and the sample chamber 8 was extrapolated. The amount of the substance to be determined in a certain amount of the sample was determined, and the detector was configured based on this and the quantitative value during the continuous analysis. The concentration of residual chlorine thus obtained was stable at 0.75 to 0.90 ppm. In parallel, the difference from the quantitative value obtained by the ortho-tolidine method is as small as 5 to 8%,
It was reliable. The detector may be installed in any direction, horizontal or vertical, and the analysis value did not change depending on the installation direction. In this embodiment,
By keeping the detector warm and constant, the reliability of the analysis could be further improved.

Example 5 Ozone in gas was continuously analyzed under the same conditions as in Example 4 except that a fluororesin thin film was used as the permeable membrane 7 and a pH 7 aqueous sodium phosphate solution containing potassium iodide was used as the electrolytic solution. Analysis results ranged from 0.1 ppm to 100 ppm
% Reproducibility. Absorption with neutral potassium iodide and the difference from the quantitative results obtained by titration were within 10%.

According to the present embodiment, a target component in a fluid can be monitored as a sensor.

Example 6 The basis weight of the detection electrode was changed from the conventional 400 gm ^-2 to 620 gm ^-2, and 1M-KCl and 1M-KH ₂ PO ₄ aqueous solution were used as the impregnation solution of the detection electrode. Similarly, the dissolved oxygen in pure water degassed with argon gas was measured. The degassing time with argon gas is 30 minutes, 45 minutes, and 60 minutes, respectively, and each sample is sent to the detector at about 5 ml / min, and the sample chamber is considered to have been sufficiently replaced with new sample. (2
Min), the liquid supply was stopped, and the current which decreased immediately after that was observed. Dissolved oxygen was determined from the extrapolated value of the iQ plot obtained by observation for 3 minutes, the dissolved oxygen after 30 minutes of deaeration was 16 ppb, the dissolved oxygen after 45 minutes of deaeration was 9 ppb, and the deaeration was 6
The dissolved oxygen at 0 minutes was 5 ppb. In parallel quantification by the dithizone method (colorimetric method), the dissolved oxygen after 30 minutes, 45 minutes and 60 minutes after degassing was about 20 ppb and about 10 ppb, respectively.
And less than 10 ppb.

〔The invention's effect〕

ADVANTAGE OF THE INVENTION According to this invention, a to-be-quantitative substance in a gas sample or a liquid sample can be quantified accurately in a short time.
Further, the detector for electrical analysis used in the present invention can be used, for example, as a high-sensitivity sensor for clinical examination.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram showing an example of a detector for electric analysis used in the present invention, and FIG. 2 is a diagram schematically showing a flow path of a sample in a sample chamber of the electric analysis detector used in the present invention. . 1 ... diaphragm, 2 ... detection electrode, 4 ... counter electrode, 7 ... transmission membrane, 8 ... sample chamber, 9 ... capillary.

Claims (4)

(57) [Claims] 1. A detection electrode chamber having a detection electrode made of a conductive porous body and a counter electrode chamber having a counter electrode are electrolyzed on a porous body of the detection electrode chamber in an adjacent electrolytic cell via an ion-permeable diaphragm. By impregnating the liquid, supplying a fixed amount of sample that has passed through the permeable membrane and electrolyzing it, and measuring at least one of the voltage, current or electric quantity at the detection electrode,
An electroanalytical method characterized by absolutely quantifying an analyte in a sample. 2. A detection electrode room having a detection electrode and a game room having a game are adjacent to each other via a diaphragm as the electrolytic cell, and the detection is carried out with a permeable membrane interposed between sample supply surfaces of the detection electrode room. The method according to claim 1, wherein a detector for electroanalysis having a sample chamber for holding a sample, which is provided on a side opposite to the pole chamber, is used. 3. A sample is continuously supplied to the detection electrode chamber through a permeable membrane provided on a sample supply surface of the detection electrode chamber, and a substance to be determined in the sample is continuously analyzed, and at any time during the continuous analysis. The supply of the sample is stopped at a current based on this,
3. The electroanalytical method according to claim 1, wherein the change in the amount of electricity or the voltage is measured, and the measured value during continuous supply is calibrated by determining the concentration of the substance to be determined. 4. An electrolysis solution according to claim 1, wherein said electrolysis solution contains a mediator for converting a component to be determined in a sample into an electrode reaction active substance. Analysis method.